Protein folding and aggregation in vitro and in vivo

Proteins are biological macromolecules that typically fold into a specific three-dimensional structure. Misfolding of proteins can cause lack of function and may lead to formation of aggregates. Protein misfolding and aggregation are associated with many human diseases, and are particularly common in neurodegenerative disorders and dementias including Alzheimer's disease, Parkinson's disease and prion diseases. In addition, formation of aggregates during the production or storage or protein-based pharmaceuticals can render the products inactive or potentially harmful. An increased understanding of the factors that control protein folding is necessary in order to develop strategies to prevent misfolding and aggregation. The ideal strategies to control folding and aggregation may differ significantly between the in vitro environment, where the biophysical and biochemical properties of the protein are the dominant factors in determining protein conformation, and the in vivo environment, where cellular control mechanisms play an active role. This thesis explores protein folding and aggregation in an in vitro system using a model beta-helical protein and an in vivo system using the microtubule binding protein, tau.;In vitro folding and aggregation studies were done using the isolated beta-helical domain of P22 tailspike protein (bhx) as a model system. Refolding of bhx from a urea-denatured state was studied using stopped-flow fluorescence. These studies showed that bhx folds via two parallel pathways where one pathway includes a slow refolding step that could be attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments with short delay times. However, double-jump refolding experiments with delay times longer than 100 seconds along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions.;Aggregation of bhx can also be induced by exposure to elevated temperature or moderate concentrations of denaturant. At temperatures near 40°C, bhx forms large aggregates, which are initially soluble and later precipitate. The rate of monomer loss under these conditions was shown to be strongly dependent on temperature, but, surprisingly, was not dependent on initial protein concentration over the range of concentrations studied (0.2 to 1 mg/mL). The lack of concentration dependence may be partially attributed to native oligomerization of bhx. When bhx is aggregated in the presence of moderate concentrations of urea, the rate of monomer loss is dependent on protein concentration, which suggests that native associations may be disrupted by urea. Finally, aggregation of bhx at low pH resulted in decreased monomer loss rates in combination with decreased aggregate solubility.;In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the Unfolded Protein Response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine if a direct mechanistic link can be demonstrated between tau aggregation and the UPR. The first model system used was SH-SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility or phosphorylation were observed. In the second model system, wild-type tau and a tau variant with increased aggregation propensity, P301L were heterologously overexpressed in HEK cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR.